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Abstract Background To this day, the effects of sodium hypochlorite and chelating agents on the smear layer and on the dentine’s surface, remain not fully examined. The study is aimed to analyze the dentine's surface treated with 40% citric acid and 5.25% sodium hypochlorite according to two irrigation protocols. Materials and methods The study employed a computer-controlled Nikon Eclipse LV100 optical microscope. Ten roots split longitudinally with canals prepared mechanically using the MTwo system to a size of 40/04 were observed. The root halves were divided into two study groups, one half in each of the groups. According to two irrigation protocols, the dentine's surface was irrigated with 40% citric acid and 5.25% sodium hypochlorite, separated with water. Dentine surface was observed in a fixed place and photographed in 500 × magnification after each irrigation stage. The obtained images were then analyzed using computer software (NIS-Elements AR, GIMP-2.6, ImageJ 1.45s). Results Various speed of removal of the smear layer and varied morphological changes of the dentine's surface were observed in both examined groups. Double irrigation of the dentine with citric acid for 30 s resulted in complete removal of the smear layer, and double irrigation for 60 s resulted in increased mean diameter of the dentine tubules (degradation of the dentine's surface). Conclusions Double alternating irrigation with citric acid and sodium hypochlorite for 30 s yielded satisfactory results, while double irrigation for 60 s resulted in increased mean diameter of the dentine tubules. A real time assessment of the dentine’s surface changes after both tested solutions can improve understanding of the phenomena that occur during the irrigation and as a result it might help to improve clinical outcomes in root canal treatment.